Clavulanic acid amine salts, their preparation and use

ABSTRACT

This invention provides a novel process for the preparation of clavulanic acid and pharmaceutically acceptable salts and esters thereof which process utilizes the preparation of the tertiary-butylamine salt of clavulanic acid optionally in the form of an acetone solvate and subsequently converting said salt into the desired product.

CROSS REFERENCE

This is a continuation of Ser. No. 479,952 filed Mar. 29, 1983, nowabandoned, which is a division of Ser. No. 334,438 filed Dec. 24, 1981now U.S. Pat. No. 4,454,069 which is a continuation of Ser. No. 179,760filed Aug. 20, 1980, abn.

This invention relates to a novel process for the preparation ofclavulanic acid of the formula (I): ##STR1## and pharmaceuticallyacceptable salts and esters thereof.

British Pat. No. 1508977 discloses inter alia that salts of clavulanicacid can be obtained by absorbing the clavulanate anion in filteredbroth on to an anion exchange resin, eluting therefrom with anelectrolyte, desalting the resulting solution, applying the desaltedsolution to a further anion exchange resin, chromatographically elutingtherefrom with an electrolyte, desalting the resulting solution andthereafter removing the solvent. This process can be used to giveacceptable yields of pure material but the use of resin columns involvessignificant investment and they can introduce limitations in large scaleproduction operations, and so it would be desirable to have analternative procedure available that involved few resin utilizingstages. British Pat. No. 1543563 discloses a process for the preparationof clavulanic acid salts via precipitation of lithium clavulanate.

It has now been found that the tertiary-butylamine salt of clavulanicacid which can be obtained in high purity is a useful intermediate inthe preparation of clavulanic acid. The salt has been disclosed inBelgian Pat. No. 862211, but only as a suitable ingredient forpharmaceutical formulations.

The present invention provides the use of the tertiary-butylamine saltof clavulanic acid as an intermediate in the preparation of clavulanicacid and pharmaceutically acceptable salts and esters thereof.

In another aspect the present invention provides a process for thepreparation of clavulanic acid or a pharmaceutically acceptable salt orester thereof which process comprises converting the tertiary-butylaminesalt of clavulanic acid into clavulanic acid or a pharmaceutically saltor ester thereof.

In a further aspect the present invention provides a process for thepurification of clavulanic acid or a pharmaceutically acceptable salt orester thereof which process comprises:

(i) contacting impure clavulanic acid in organic solvent withtertiary-butylamine,

(ii) isolating the tertiary-butylamine salt of clavulanic acid, and

(iii) converting the thus formed tertiary-butylamine salt intoclavulanic acid or a pharmaceutically acceptable salt or ester thereof.

The pharmaceutically acceptable salts and esters of clavulanic acidprepared by the processes of this invention are those described inBritish Patent Specification Nos. 1508977 and 1508978 which are hereinincorporated by reference.

Particularly suitable salts include the pharmaceutically acceptablealkali and alkaline earth metal salts, for example the sodium,potassium, calcium and magnesium salts. Of these salts the sodium andpotassium are most suitable and the potassium is preferred.

Suitable esters include those cleavable to provide clavulanic acid or asalt thereof, by chemical methods such as hydrogenolysis or bybiological methods.

Suitably the carboxylic acid is esterified by a group of the sub-formula(a), (b), (c) or (d): ##STR2## wherein R¹ ia a hydrogen atom or analkyl, alkenyl or alkynyl group of up to 3 carbon atoms; R² is ahydrogen atom or a methyl group; R³ is a phenyl group or a phenyl groupsubstituted by a fluorine, chlorine or bromine atom or a nitro, methylor methoxy group; R⁴ is a hydrogen atom or a phenyl group or a phenylgroup substituted by a fluorine, chlorine or bromine atom or a nitro,methyl or methoxy group; R⁵ is a hydrogen atom or a methyl group; R⁶ isa C₁₋₄ alkyl, phenyl or C₁₋₄ alkoxy group or R⁵ is joined to R⁶ to forma phthalidyl, dimethylphthalidyl or dimethoxyphthalidyl group; and R⁷ isa C₁₋₄ alkyl, phenyl, chlorophenyl or nitrophenyl group; or CHR¹ R² is aphenacyl or bromophenacyl group.

Favourably R¹ is a hydrogen atom or a methyl, ethyl, vinyl or ethenylgroup. Favourably R² is a hydrogen atom. Favourably R³ is a phenyl,p-bromophenyl, p-methoxyphenyl or p-nitrophenyl group. Favourably R⁴ isa hydrogen atom. Favourably R⁶ is a methyl, t-butyl or ethoxy group oris joined to R⁵. Favourably R⁷ is a methyl group.

Preferred groups of the sub-formula (a) include the methyl and ethylgroups.

Preferred groups of the sub-formula (b) include the benzyl andp-nitrobenzyl groups.

Preferred groups of the sub-formula (c) include the acetoxymethyl,pivaloyloxymethyl, α-ethoxycarbonyloxymethyl and phthalidyl groups.

A preferred group of the sub-formula (d) is the methoxymethyl group. Thesource of impure clavulanate material for use in this process may be anyclavulanate producing micro-organism; liquors obtained by formentationusing such micro-organisms may be treated in conventional manner priorto solvent extraction, for example as described in British PatentSpecification No. 1508977.

The tertiary-butylamine salt of clavulanic acid is obtained bycontacting tertiary-butylamine (2-amino-2-methylpropane) with theclavulanic acid. This is conveniently performed by adding a solution oftertiarybutylamine in an organic solvent to a solution of impureclavulanic acid in an organic solvent.

The solution of clavulanic acid in organic solvent may be obtained byextraction of an acidified aqueous solution of clavulanic acid.Preferably the pH of the aqueous solution prior to extraction is in therange 1 to 3. Preferably the extraction is carried out at a temperaturefrom 5° to 15° C. The aqueous solution should contain at leastapproximately 25 mg/ml, preferably approximately 100 mg/ml of clavulanicacid to give best results. The aqueous acidified solution for extractionmay be conveniently obtained from a fermentation broth by acidifying anaqueous solution obtained by absorbing the clavulanate anion from thebroth onto an anion exchange resin, eluting the clavulanate therefromwith an aqueous solution of an electrolyte, and optionally desalting.

Suitable organic solvents in which impure clavulanic acid is contactedwith tertiary-butylamine include those of the formula (II): ##STR3##wherein R⁸ is a C₁₋₆ alkyl group or a C₁₋₆ alkoxy group and R⁹ is a C₁₋₆alkyl group. Further suitable solvents include ethers such astetrahydrofuran and dioxan. The present invention also encompassesmixtures of such solvents.

More suitably the organic solvent is one which can be used directly toextract the acidified aqueous solution and may be selected from thegroup consisting of ethyl acetate, methyl acetate, propyl acetate, nbutyl acetate, methyl ethyl ketone, methyl isobutyl ketone,tetrahydrofuran and mixtures of such solvents. Of these the mostsuitable are methyl isobutyl ketone, methyl ethyl ketone, and ethylacetate. Suitable solvent mixtures include methyl ethyl ketone/methylisobutyl ketone and tetrahydrofuran/methyl isobutyl ketone. A preferredsolvent is ethyl acetate.

Suitable solvents for tertiary-butylamine include acetone, ethylacetate, methyl isobutyl ketone, and methyl ethyl ketone. Of theseacetone is preferred.

It has been found that the process is operable in presence of a smallamount of water in the solvent or solvent mixture. Typically 0-7%(vol/vol) water may be present, preferably less than 4%. More suitablyhowever the solution of the clavulanic acid is subjected to drying, forexample over magnesium sulphate.

In general one equivalent of tertiary-butylamine or a slight excessthereof is used to produce the salt of clavulanic acid. The solutionsare generally mixed slowly with stirring and the mixture stirred forsome time after addition is complete. The desired tertiary-butylaminesalt of clavulanic acid may then be isolated. In this way thetertiary-butylamine salt of clavulanic acid is separated from most orall of the impurities. This may be effected in conventional manner, forexample by centrifugation with subsequent removal of the supernatantliquid.

In one preferred embodiment of this invention, the tertiary-butylamineclavulanate is employed as an acetone solvate. This acetone solvate hasadvantageous stability and purity characteristics compared to previouslyknown forms of the tertiary-butylamine salt of clavulanic acid, see forexample Belgian Pat. No. 862211. The solvate is particularly useful inthe present invention because it can readily be isolated as a highlypure and stable crystalline compound.

Accordingly the present invention also provides the tertiary-butylaminesalt of clavulanic acid in the form of an acetone solvate.

In the presence of acetone, the tertiary-butylamine salt of clavulanicacid precipitates in the form of an acetone solvate, comprising boundacetone at levels corresponding to hemi- or mono-solvation. Duringisolation and/or drying, some acetone may be lost since the strength ofsolvation is not high, but the amount of acetone in the product is notcritical and in general varies up to 17% (w/w) (that is to say up tomono-solvation). We have found that the percentage of acetone in thesolvate is suitably between 2%-9%, more suitably between 4%-8%, andpreferably about 7%. It has been found that the percentage of acetone inthe solvate may be greatly diminished by washing with organic solvent,for example methyl isobutyl ketone or ethyl acetate. However, it isdifficult to remove the acetone content under vacuum indicating that adistinct chemical entity is present. The acetone solvate is formed bycontacting clavulanic acid in organic solvent with tertiary-butylaminein the presence of acetone.

In general, a solution containing clavulanic acid is mixed with at leastthe same volume of acetone together with the tertiary-butylamine, whenthe salt is precipitated.

Preferably tertiary-butylamine dissolved in acetone is mixed with asolution of clavulanic acid in an organic solvent. Favoured organicsolvents include ethyl acetate, tetrahydrofuran, methyl ethyl ketone,methyl isobutyl ketone and mixtures of such solvents, of which ethylacetate is preferred. It is preferred that in order to form the desiredacetone solvate the organic solvent:acetone ratio after mixing should beapproximately 1:1.

Recrystallisation of the initial acetone solvate is often advantageousto further reduce the level of impurities. A convenient solvent for therecrystallisation is aqueous acetone. Such recrystallisation isperformed in conventional manner, for example the solvate is dissolvedin water, treated with a small amount of acetone, filtered, and thentreated with larger volumes of acetone optionally with stirring and/orcooling to afford the recrystallised product.

The tertiary-butylamine salt of clavulanic acid optionally in the formof its acetone solvate may be converted into clavulanic acid or apharmaceutically acceptable salt or ester thereof by ion-replacement orby esterification.

Such ion-replacement may be performed using ion-exchange resins, forexample by passing a solution of the tertiary-butylamine salt through abed of a cation exchange resin in sodium, potassium or calcium form.

Suitable cation exchange resins include Amberlite IR 120 and equivalentresins. Alternatively ion-replacement may be effected by metathesis ofthe tertiary-butylamine cation with a base, for example a carbonate,bicarbonate or hydroxide or pharmaceutically acceptable alkali oralkaline earth metal, or a salt of an organic carboxylic acid, forexample a salt of an alkanoic acid of formula (III):

    R.sup.10 --CO.sub.2 H                                      (III)

wherein R¹⁰ is an alkyl group, containing for example from 1 to 20carbon atoms, preferably from 1 to 8 carbon atoms. Examples of suitablesalts include the acetate, propionate or ethylhexanoate salts, potassium2-ethylhexanoate and sodium 2-ethylhexanoate being preferred.

Suitable methods of esterification include:

(a) the reaction of the tertiary-butylamine salt of clavulanic acid witha compound of the formula Q-R¹¹ wherein Q is a readily displaceablegroup and R¹¹ is an organic group;

(b) the reaction of the tertiary-butylamine salt of clavulanic acid withan alcohol or thiol in the presence of a condensation promoting agentsuch as carbodi-imide; and

(c) the reaction of the tertiary-butylamine salt of clavulanic acid witha diazo compound.

It is of course realised that the foregoing processes extend to coverthose aspects wherein the tertiary-butylamine salt is first converted toclavulanic acid or other salt and subsequently is converted to thedesired ester.

Further details of esterification methods are disclosed in BritishPatent Specification Nos. 1508977 and 1508978.

Use of the present invention enables salts and esters of clavulanic acidto be more readily obtained in pure form than operation of the processesof British Patent Specification Nos. 1508977 and 1543563. For example,one preferred product, potassium clavulanate, can be prepared incrystalline form by the methods of said patents but these crystals aremicrocrystals. However the present invention enables potassiumclavulanate to be produced in pure form in large crystals which may bewell-defined needles or waisted plates (i.e. butterfly-shaped).

The following Examples illustrate the invention:

EXAMPLE 1

An ethyl acetate extract obtained by known methods containing crudeclavulanic acid (68 l assayed at 24.75 mg/ml) was stirred for 10 minuteswith MgSO₄ (1.7 kg). Carbon (1.7 kg "Norrit GSX") was added and themixture stirred for a further 10 minutes. The slurry was filtered bysucking through a fine-woven canvas filter (a Nutsche filter) which wasthen washed with ethyl acetate (10.1). The combined filtrate and washing(71 l total) contained 23.7 mg/ml of clavulanic acid in dry ethylacetate. To this was added acetone (71 l) and then a solution oft-butylamine (1.13 l) in acetone (5.6 l) over 30 minutes. The mixturewas stirred for 60 minutes and then the solid product separated bycentrifugation and then washed with acetone (2×10 l) and finally driedon a fluid bed drier (ambient temperature) to yield the t-butylaminesalt of clavulanic acid acetone solvate (2.25 kg), assaying at 65.8%free acid and 97% as T.B.A. clavulate acetone solvate. Yield correctedfor purity, 88%. Infra-red (Nujol Mull) 1780 (β-lactam carbonyl), 1708(solvated acetone), 1695 cm⁻¹ (C═C). For comparison addition of freeacetone to the above sample gave rise to an absorption at 1721 cm⁻¹corresponding to free acetone, and the non-solvated t-butylamine salt ofclavulanic acid shows no absorption at 1708 cm⁻¹.

EXAMPLE 2 Recrystallisation of t-butylamine Clavulanate Acetone Solvate

A sample of the product of Example 1 was dissolved in water to yield a20% w/v solution (based on clavulanate ion) at ambient temperature.Acetone (8 vols) was added over about 10 minutes and the resultingsolution filtered. To the filtrate is added further acetone (32 vols)over about 30 minutes and the resulting mixture stirred for a further 2hours with chilling to 0°-5° C. The precipitate was filtered off anddried in a fluid bed drier to yield the t-butylamine salt of clavulanicacid acetone solvate. (assay 66.0% free acid, 99.7% T.B.A. clavulanateacetone solvate. Yield, corrected for purity, 85%). (The product of thisExample was about 1-2% purer than the starting material).

EXAMPLE 3

t-Butylamine salt of clavulanic acid acetone solvate (2.55 kg) wasdissolved in isopropanol (68 l) containing water (1.4 l) at 20° C. Thesolution was filtered through an in-line filter and washed through withisopropanol (2.1 l). Potassium ethyl hexanoate in isopropanol (1.4equiv., 6.1 l of 2N solution) was added over 20 minutes. The resultingmixture was stirred for 30 minutes at ambient temperature and chilledfor 2 hours to 0°-5° C. The product was filtered by suction through afine-woven canvas filter (Nutsche) with nitrogen purge, washed withisopropanol (10 l) and acetone (10 l). The product was then dried undervacuum at ambient temperature to yield potassium clavulanate (1.8 kg).Assay 83.2%. Yield, corrected for purity, 89%.

EXAMPLE 4

The tertiary-butylamine salt of clavulanic acid (non-solvated) (28.7 g,69.8% as pure free acid) was dissolved in isopropanol (760 ml)containing water (10-40 ml) at 20° C. The solution was filtered andwashed through with further isopropanol (25 ml). Potassium ethylhexanoate in isopropanol (71 ml of 2N solution, 1.4 equivalents) wasadded over 15 minutes. The resulting mixture was stirred for 30 minutesat ambient temperature and then chilled for 2 hours at 0°-5° C. Theproduct was filtered, washed with isopropanl (100 ml) and acetone (100ml). The product was then dried under vacuum at ambient temperature toyield potassium clavulanate (20.5 g, 81.2%), yield 83.0%.

What is claimed is:
 1. The tertiary-butylamine salt of clavulanic acidin the form of an acetone solvate.
 2. The salt of claim 1 having 4%-8%(w/w) acetone.
 3. The salt of claim 1 having about 7% (w/w) acetone.